A SNP molecular marker related to the number of intermuscular bones of yellow river carp, a primer set and application in breeding less intermuscular spines of yellow river carp

Abstract

The present application belongs to the field of aquatic genetic breeding, and relates to a SNP molecular marker related to the number of intermuscular bones of yellow river carp, primer groups and application in breeding yellow river carp with less intermuscular spines; the present application provides a group of SNP molecular markers significantly related to the number of intermuscular bones of yellow river carp, and the yellow river carp with less number of intermuscular spines can be quickly screened by using the group of SNP molecular markers. Based on the phenotype determination of the number of intermuscular bones of 917 yellow river carps and the whole genome resequencing data of 445 individuals, through GWAS analysis, the present application first identifies the SNP site on chromosome A24 which is significantly related to the number of intermuscular bones, and provides a reliable molecular marker for the breeding of low intermuscular bone strain of yellow river carp.

Description

Technical Field

[0001] This invention belongs to the field of aquatic genetics and breeding, and relates to the breeding of Yellow River carp with fewer intermuscular spines. Background Technology

[0002] The Yellow River carp (Cyprinus carpio haematopterus) belongs to the order Cypriniformes, family Cyprinidae, and genus Cyprinus. It is an important freshwater economic fish in my country and a local specialty species formed through natural selection in the Yellow River basin. It is characterized by golden scales and red tails, a long, spindle-shaped body, and tender flesh. It also has a wide diet, strong disease resistance, and resilience. Large-scale Yellow River carp farming is widespread in cities and counties along the Yellow River, with the Yellow River carp from the Henan section of the Yellow River being particularly famous. However, with the improvement of living standards and the diversification of fish species, the Yellow River carp, due to the large number of intermuscular bones in the muscle layers on both sides of its vertebrae, has a significant impact on its edible quality and processing efficiency, posing a major challenge to the Yellow River carp industry. Therefore, breeding Yellow River carp with fewer intermuscular bones is an important direction for the sustainable development of the carp industry.

[0003] Intermuscular bones exist only in lower teleost fishes and are needle-like ossifications directly derived from mesenchymal cells in the muscle layers on both sides of the vertebrae. Based on their attachment location within the fish (medullary arch, vascular arch, and vertebral body), intermuscular bones can be classified into three types: medullary arch ossicles, vascular arch ossicles, and vertebral body ossicles. Descriptions of intermuscular bones in China began in the mid-20th century. To date, the morphology, distribution, and ossification patterns of intermuscular bones in various lower teleost fish species, including silver carp, Yellow River carp, blunt snout bream, white-lipped bream, and crucian carp, have been reported, indicating that the number of intermuscular bones is a quantitative trait controlled by multiple genes. However, due to the difficulty in measuring the number of intermuscular bones in vivo, effective selection using conventional breeding methods is not feasible. While significant progress has been made in obtaining boneless fish through gene editing, the use of gene-edited fish in aquaculture is strictly limited. Therefore, there is an urgent need to develop molecular markers related to intermuscular bones for assisted breeding to cultivate Yellow River carp with few or no intermuscular bones.

[0004] Currently, no stable SNP molecular markers have been reported for precise selection of intermuscular bone quantity in Yellow River carp. Our research group previously developed a genome-wide bioinformatics-based breeding method for carp (CN116825190A). By performing whole-genome resequencing on target individuals, a large number of SNP loci can be obtained. Through quantitative trait locus (QTL) mapping analysis or genome-wide association analysis (GWAS), SNP markers and their genotyping information related to the quantity trait of intermuscular bone in Yellow River carp can be screened. These markers can be used to select Yellow River carp individuals conforming to the target genotype as parents before breeding, thereby obtaining offspring with less intermuscular bone. Summary of the Invention

[0005] This invention proposes an SNP molecular marker and primer set related to the number of intermuscular bones in Yellow River carp, and its application in the breeding of Yellow River carp with few intermuscular bones, which can realize early and accurate selection of the trait of few intermuscular bones in Yellow River carp.

[0006] The technical solution of this invention is implemented as follows:

[0007] Firstly, regarding the SNP molecular markers related to the number of intermuscular bones in Yellow River carp, the two SNP molecular markers mentioned above are located at base 20756481 and base 20542471 of A24 in the carp genome (GenBank no: GCA_018340385.1), respectively. The SNP molecular markers located at base 20756481 of A24 in the Yellow River carp chromosome have a polymorphic base of T / C; or / and located at base 20542471 of A24 in the Yellow River carp chromosome have a polymorphic base of T / G.

[0008] Secondly, the application of the aforementioned SNP molecular markers in the breeding of Yellow River carp with few intermuscular spines.

[0009] Thirdly, the primer set for detecting the aforementioned SNP molecular markers.

[0010] The primer sets described above are nucleotide sequences as described in SEQ ID No. 1 and SEQ ID No. 2 or nucleotide sequences as described in SEQ ID No. 3 and SEQ ID No. 4.

[0011] Fourthly, the aforementioned product for detecting the number of intermuscular bones in Yellow River carp includes the primer set described above. It also includes other reagents for PCR amplification.

[0012] Fifthly, the application of the aforementioned SNP molecular markers in the breeding of Yellow River carp with few intermuscular spines involves the following steps:

[0013] (1) Extract genomic DNA from individual Yellow River carp to be tested;

[0014] (2) Using genomic DNA as a template, the SNP molecular marker sites described in claim 1 are amplified, and the amplification products are sequenced to obtain the genotype of the SNP molecular markers of the Yellow River carp to be tested;

[0015] (3) Based on the obtained genotype, screen Yellow River carp with less intermuscular bone to determine whether the Yellow River carp to be tested has the potential for the quantitative trait of less intermuscular bone.

[0016] The SNP marker located at position 20756481 on chromosome A24 was detected. Yellow River carp with genotype CC exhibited the trait of having fewer intermuscular bones. The SNP marker located at position 20542471 on chromosome A24 was detected. Yellow River carp with genotype GG exhibited the trait of having fewer intermuscular bones.

[0017] Two SNP loci were detected simultaneously, and only homozygous individuals with genotypes CC and GG were retained as parents. Through continuous multi-generational selection, a stable strain of Yellow River carp with less intermuscular bone was obtained.

[0018] The present invention has the following beneficial effects:

[0019] 1. This invention provides a set of SNP molecular markers significantly associated with the number of intermuscular bones in Yellow River carp. These SNP markers can be used to rapidly screen Yellow River carp with fewer intermuscular bones. Based on phenotypic analysis of the number of intermuscular bones in 917 Yellow River carp and whole-genome resequencing data from 445 individuals, this invention, through GWAS analysis, identifies for the first time an SNP locus located on chromosome A24 that is significantly associated with the number of intermuscular bones, providing reliable molecular markers for the breeding of Yellow River carp strains with low intermuscular bone counts.

[0020] 2. Based on genome-wide association analysis (GWAS) screening, the marker genetic effects are clear, with a single locus achieving 7.7%-8.8% phenotypic variation, resulting in high selection accuracy. This invention provides a method for cultivating new strains of Yellow River carp with less intermuscular bone, enabling early selection, shortening the breeding cycle, and improving the edible quality and economic value of commercial fish. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The results show the correlation analysis between SNP markers and the total number of intermuscular bones; where A is the Manhattan plot and B is the QQ plot.

[0023] Figure 2 Box plots showing the total number of intermuscular bones for different genotypes of SNP A24:20756481, where A represents the experimental population and B represents the validation population.

[0024] Figure 3 Box plots are shown for the total number of intermuscular bones in different genotypes of SNP A24:20542471, where A represents the experimental population and B represents the validation population.

[0025] Figure 4 The target fragment sequence contains SNP A24:20756481.

[0026] Figure 5 The target fragment sequence contains SNP A24:20542471. Detailed Implementation

[0027] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0028] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0029] Example: Screening of SNP molecular markers

[0030] 1. Extract genomic DNA from individual Yellow River carp to be tested.

[0031] The study included 52 full-sib families of Yellow River carp. At 8 months of age, 445 fish were randomly selected and euthanized using methanesulfonate (MS-222). The number of intermuscular bones in each fish was recorded through dissection. Genomic DNA was extracted from the fin rays of the Yellow River carp using a standard DNA extraction kit. DNA purity was assessed on a 1% agarose gel, and DNA concentration was measured using a Qubit 4.0 fluorometer (Thermo Scientific).

[0032] 2. SNP molecular marker genotyping of individual Yellow River carp.

[0033] Genome resequencing was performed using the DNBSEQ-T7 platform. Reads from each sample were aligned with the latest carp reference genome (GenBank ID: GCA_018340385.1) using BWA v0.7.17. After alignment, SNP variant detection was performed using GATK v4.0. Then, using the "-geno", "-hwe", and "-maf" parameters in PLINK v1.90, SNP markers with genotype loss rates greater than 2%, Hardy-Weinberg equilibrium less than 1.0e-6, and minimum allele frequencies less than 5% were removed from all individuals. Individuals with genotype loss rates less than 5% were retained. Genome-wide SNP annotation was performed using ANNOVAR. Missing genotypes were filled using BEAGLE 5.0.

[0034] 3. Genome-wide association analysis (GWAS) of intermuscular bone quantity trait

[0035] Genome-wide association analysis (GWA) of the intermuscular bone quantitative trait in Yellow River carp was performed using a linear mixture model of GEMMA. A conservative Bonferroni correction was used to determine the genome-wide significance threshold (P = 0.05 / N), where N is the number of independently segregating SNPs, calculated using Plink v.1.9 software. SNPs with P values ​​exceeding the threshold were considered significantly associated with the intermuscular bone quantitative trait in Yellow River carp. QQ plots and Manhattan plots were constructed using the ggplot2 package in R. QQ plots were used to determine the validity of P values ​​in the genome-wide association study, and Manhattan plots were used to visualize the results of genome-wide P values.

[0036] 4. Correlation analysis between SNP molecular marker genotype and intermuscular bone quantity.

[0037] A total of 4,854,366 SNP loci were retained for GWAS analysis. The genome-wide significance threshold was 1.03 × 10⁻⁶. -8 (0.05 / 4854366). GWAS results for the quantitative traits of intermuscular bones in Yellow River carp are as follows: Figure 1 As shown (where Figure 1 A is a Manhattan diagram representing the total number of intermuscular bones. Figure 1 B is the QQ plot of the total number of intermuscular bones: Two SNPs significantly associated with the number of intermuscular bones in Yellow River carp at the genomic level were detected on chromosome A24. These are A24:20756481 and A24:20542471. Among them, the SNP with the highest significance in the total number of intermuscular bones (A24:20756481) explained 8.88% of the phenotypic variation (PVE). SNP (A24:20542471) was significantly associated with the total number of intermuscular bones (p = 6.28e-09, PVE = 7.7%).

[0038] The total number of intermuscular bones in individuals carrying different genotypes of SNP (A24:20756481) is as follows: Figure 2 As shown in Figure A, there were significant differences in the average total number of intermuscular bones among the three genotypes at the SNP (A24:20756481) locus in Yellow River carp. Among them, the average total number of intermuscular bones in Yellow River carp carrying the TT genotype was significantly higher than that of the TC and CC genotypes.

[0039] The total number of intermuscular bones in individuals carrying different genotypes of SNP (A24:20542471) is as follows: Figure 3 As shown in Figure A, there were significant differences in the average total number of intermuscular bones among the three genotypes at this locus. Specifically, the average total number of intermuscular bones in Yellow River carp carrying the TT genotype was significantly higher than that of the TG and GG genotypes.

[0040] Application example: Validation of SNP molecular markers related to the number of intermuscular thorns

[0041] Thirty-five Yellow River carp were randomly selected. Through dissection and separation, the total number of intermuscular bones in each fish was counted. Simultaneously, tail fin samples were collected from each fish for DNA extraction, following the same procedure as in Example 1. Using the aforementioned DNA as a template, PCR amplification was performed using SEQ ID NO:1 and SEQ ID NO:2 as primers to obtain gene fragments containing SNPs A24:20756481 and A24:20542471.

[0042] The sequences corresponding to the upstream and downstream primers at each locus are as follows:

[0043]

[0044] This includes a gene fragment containing SNP A24:20756481, such as Figure 4 As shown.

[0045] This includes a gene fragment containing SNP A24:20542471, such as Figure 5 As shown.

[0046] Sanger sequencing was used to obtain the genotype of each individual, and the total number of intermuscular bones in individuals with different genotypes was counted. In the validation population, SNP A24:20756481 was detected with two genotypes: CC and TC. Figure 2 B presents the validation results for SNP A24:20756481. (By...) Figure 2 B indicates that there are significant differences in the total number of intermuscular bones among individuals with different genotypes of Yellow River carp SNP A24:20756481; among them, individuals carrying the CC genotype have a smaller total number of intermuscular bones than individuals carrying the TC genotype. (Compared to Example 1) Figure 2 Result A is consistent.

[0047] In the validation population, SNP A24:20542471 was detected with two genotypes: GG, TG, and TT. Figure 3 B presents the verification results for SNPA24:20542471. (By...) Figure 3 B indicates that there are significant differences in the total number of intermuscular bones among individuals with different genotypes of Yellow River carp SNP A24:20542471; among them, individuals carrying the GG genotype have a smaller total number of intermuscular bones than individuals carrying the TG and TT genotypes. (Compared to Example 1) Figure 3 Result A is consistent.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. SNP molecular markers associated with the number of intermuscular bones in Yellow River carp, characterized in that, The SNP molecular marker is located at the 20756481st base of chromosome A24 of the Yellow River carp, with a polymorphic base of T / C; or / and located at the 20542471st base of chromosome A24 of the Yellow River carp, with a polymorphic base of T / G.

2. The SNP molecular marker related to the number of intermuscular bones in Yellow River carp according to claim 1, characterized in that: The Yellow River carp chromosome was selected from the carp genome in GenBank no: GCA_018340385.

1.

3. The application of the SNP molecular marker as described in claim 1 or 2 in the breeding of Yellow River carp with few intermuscular spines.

4. A set of primers for detecting the SNP molecular markers of claim 1 or 2.

5. The primer set according to claim 4, characterized in that: The primer set is a nucleotide sequence as described in SEQ ID No. 1 and SEQ ID No. 2 or a nucleotide sequence as described in SEQ ID No. 3 and SEQ ID No.

4.

6. A product for detecting the number of intermuscular bones in Yellow River carp, characterized in that: The product includes the primer set as described in claim 4.

7. The product for detecting the number of intermuscular bones in Yellow River carp according to claim 6, characterized in that: It also includes other reagents for PCR amplification.

8. The application of the SNP molecular marker as described in claim 1 in the breeding of Yellow River carp with few intermuscular spines, characterized in that, The steps are as follows: (1) Extract genomic DNA from individual Yellow River carp to be tested; (2) Using genomic DNA as a template, the SNP molecular marker sites described in claim 1 are amplified, and the amplification products are sequenced to obtain the genotype of the SNP molecular markers of the Yellow River carp to be tested; (3) Based on the obtained genotype, screen Yellow River carp with less intermuscular bone to determine whether the Yellow River carp to be tested has the potential for the quantitative trait of less intermuscular bone.

9. The application of the SNP molecular marker according to claim 8 in the breeding of Yellow River carp with few intermuscular spines, characterized in that: The SNP molecular marker located at the 20756481st base of chromosome A24 was detected. Yellow River carp with the genotype CC have the trait of having fewer intermuscular bones. The SNP molecular marker located at base 20542471 on chromosome A24 was detected. Yellow River carp with genotype GG have the trait of low number of intermuscular bones.

10. The application of the SNP molecular marker according to claim 8 in the breeding of Yellow River carp with few intermuscular spines, characterized in that: Two SNP loci were detected simultaneously, and only homozygous individuals with genotypes CC and GG were retained as parents. Through continuous multi-generational selection, a stable strain of Yellow River carp with less intermuscular bone was obtained.

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